The proposed studies are designed to characterize mechanism(s) of septic shock, which still remains a major cause of morbidity and mortality in the United States. The dysregulated response of innate immunity caused by bacterial infections is responsible for the clinical manifestation of septic shock syndrome. This immune response leads to upregulation of host defense but, unfortunately, the same response also causes cellular injury and septic shock. The PI and his colleague plan to use immunologic, molecular biological and biochemical approaches to investigate mechanisms associated with activation of the innate immunity by bacterial products. LPS will be used as a prototype of bacterial toxins in order to characterize the functional role of the components of LPS receptor complex (LPSRC), e.g. CD14 and Toll-like receptors (TLRs), in activation of myeloid lineage cells. To address this question, quantitative LPS binding assays and other biochemical techniques will be used to define the sequence of events occurring at the cell surface after LPS binds to CD14. Specifically, they will attempted to identify which, if any, candidate protein within the complex (TLRs, MD-2 etc.) binds LPS and whether LPS binding causes composition changes in the LPSRC. Intracellular signaling events induced by ligation of LPSRC will be characterized with a specific focus on upstream components that link LPSRC to the p38 and JNK kinase pathways. The results of in vitro studies will be used to guide the proposed in vivo studies to investigate the functional role of LPSRC in a rabbit model of endotoxic shock that closely resembles septic shock in humans. To accomplish the proposed in vivo studies, the PI and his colleagues suggest the generation of a panel of monoclonal antibodies against different components of rabbit LPSRC. The results of these studies, if successfully accomplished, may lead to the development of new strategies to control the innate immune responses in bacterial infection.